The overall goal of this project is the development and in vivo testing of a set of contrast agents that will allow noninvasive imaging of neuronal calcium levels by MRI. These reagents will provide a rapid, direct, and potentially cellular-resolution readout of dynamic brain activity. The new MRI calcium sensors will have a great impact on brain research, both through applications to the study of neurological disease in animal models, and as tools for the analysis of neural network function in basic neuroscience. The long-term objectives of this laboratory include applying the new sensors in transgenic rodents to dissect neural circuitry involved in learning and memory. Work performed under this proposal will establish a methodological platform for future biological studies. The contrast agents we will synthesize and test take advantage of the unique potency of superparamagnetic iron oxide nanoparticles (SPIOs) as imaging agents in MRI. Sensors are formed by conjugating calcium sensor proteins to the SPIOs; in the presence of calcium, the particles aggregate and produce large MRI intensity changes in T2-weighted images. A prototype sensor has been formed by conjugating calmodulin (CaM) and its substrate peptide M13 to two populations of nanoparticles; calcium-dependent aggregation with an EC50 of 0.8 mu/M Ca was observed along with large MRI signal changes, but some modifications of this sensor are required for calcium sensing in cells. In Specific Aim 1, we propose computational modeling and site-directed mutagenesis of the CaM/M13 interaction interface to reduce the sensor's potential for cross-reactivity with cellular proteins. In Specific Aim 2, we propose synthesis of new ultrasmall iron oxide nanoparticle conjugates (diameter<< 20 nm) which will respond quickly to changes in calcium concentration. A revision of our prototype calcium sensor that incorporates results of Aims 1 and 2 will be ideal for further studies in vivo. In Specific Aim 3, we propose to test the calcium responses of new nanoparticle sensors in cells, first by injection into Xenopus oocytes, and then by injection into blowfly neurons-previous work from our laboratory showed that the blowfly is a good test system for neuroimaging agents because of its ease of handling, large neurons, and absence of hemodynamic effects. Noninvasive delivery and applications of these proposed MRI calcium sensors in mammalian brains are beyond the scope of this proposal, but constitute a further step in our trajectory.